NMR Structure of a de Novo Potassium Channel Inhibitor

Natanel Mendelman, Department of Chemistry, Bar Ilan University, Ramat Gan, Israel
Ruiming Zhao, Department of Biochemistry, Brandeis University, Waltham, USA
Steve A. N. Goldstein, Department of Biochemistry, Brandeis University, Waltham, USA
Jordan Chill, Department of Chemistry, Bar Ilan University, Ramat Gan, Israel

Peptide toxins, typically small disulfide-bonded polypeptides, exert their biological activity by binding and inhibiting membrane-embedded ion channels that enable the selective passage of ions at a rate approaching the diffusion limit. Such toxins are widely employed to unravel the function of their target channels, and are also under scrutiny as therapeutic agents for several neurological and autoimmune diseases. Although toxins can be isolated from natural venoms or by traditional heterologous expression and purification methods, these approaches are limited and inconvenient for high throughput work. Recently, a combinatorial library containing peptide variants based on the ShK-toxin scaffold was used to discover a new channel-inhibiting toxin with nanomolar affinity. Here we present the structure determination of this de novo toxin as obtained from standard 2D NMR experiments. A total of 256 distance constraints were obtained from 2D NOESY spectra. In addition, 29 backbone dihedral angles constraints were derived from J-coupling measurements and TALOS analysis of chemical shifts, and eight χ1 angles were estimated from Hα/Hβ NOEs. These constraints were the basis for structure calculation using the CNS platform. To eliminate erroneous constraints, the calculations were performed iteratively, introducing first short-range and later long-range constraints, followed by dihedral angles. The ensemble quality of the 25 lowest energy structures was validated by Ramachandran analysis performed using the PROCHECK_NMR program. This new structure will be used for docking calculations and as a starting point for determining the structure of the inhibited channel.